Two Regions of Down-Regulation in the IgE-Mediated Signaling Pathway in Human Basophils

Abstract

Previous studies demonstrated that after stimulation of human basophils with a polyclonal anti-IgE Ab, early signaling elements showed sustained phosphorylation, whereas later elements were transient, suggesting that a region of down-regulation involved inhibition of phosphatidylinositol (PI) 3 kinase or its products. However, the current studies show that under some conditions, syk phosphorylation is transient. Generally, stimulation with a variety of Ags makes this early form of down-regulation more apparent. An exploration of the conditions needed to induce early down-regulation indicates that both the nature of aggregation and the cell surface density of IgE play roles. It was also found that the previously described late form of down-regulation (PI3 kinase product transience) can occur in cells displaying early down-regulation (transient syk phosphorylation), but this phenomenon is revealed by testing for subsequent down-regulation of the response to non-cross-reacting stimuli, altering their ability to induce phosphorylation of Akt or extracellular signal-regulated kinase. In contrast, phosphorylation of syk kinase, in response to a non-cross-reacting stimulus, was relatively unaffected by prior stimulation. The magnitude of cross-desensitization of the Akt or extracellular signal-regulated kinase response was a function of the strength of the first stimulus. Mediator release showed a similar cross-desensitization effect. Therefore, stimulation induces two forms of down-regulation, one operating before or at the level of syk phosphorylation, possibly characterizing the process formerly known as specific desensitization, and one that operates in the region of PI3 kinase, accounting for the process formerly known as nonspecific desensitization, which is dependent on the strength of stimulus.

Flow‐assisted quantification of in vitro activated basophils in the diagnosis of wasp venom allergy and follow‐up of wasp venom immunotherapy

“…From these data, different mechanisms that might lead to loss of patient's responsiveness during semirush immunotherapy have been postulated (36)(37)(38)(39)(40). As in vitro stimulation of basophils closely resembles the in vivo pathway finally leading to symptoms, we speculated that suppression of venom-induced CD63 expression during treatment might be an in vitro correlate of clinical tolerance and be useful to monitor successful VIT.…”

Antigen-Induced Reduction in Mast Cell and Basophil Functional Responses due to Reduced Syk Protein Levels

“…This acute mechanism for FcεRI downregulation does not appear to involve FcεRI/antigen internalization or Syk loss. Recent studies by MacGlashan [25 ]demonstrated that antigen stimulation of basophils induces downregulation operating before or at the level of Syk phosphorylation. In these experiments basophils were challenged with antigen for less than 2 h before challenge with anti-IgE.…”

Nonspecific Desensitization, Functional Memory, and the Characteristics of SHIP Phosphorylation following IgE-Mediated Stimulation of Human Basophils

“…In the relatively short time frame of typical experiments, down-regulation or desensitization is not the loss of cell surface receptors but instead appears to be the result of changes in the signaling apparatus (1). We have identified two regions of down-regulation occurring during IgE-mediated secretion (2). The earliest process precedes the activation of syk kinase but a second process, occurring at a point just distal to the activation of PI3K, occurs simultaneously with the receptor-proximal process (3).…”

“…In previous studies, we did not find any evidence of the interaction of the Fc portion of our goat anti-IgE Ab with Fc␥RIIb known to be present on basophils (2). However, activation of SHIP is expected by co-cross-linking of Fc␥RIIb with Fc RI (25,32,33), so we re-examined stimulation with anti-IgE Ab (0.2 g/ml) in the presence of high concentrations of nonspecific human IgG (previously shown to modify association of mIgG with Fc␥RIIb on basophils (2)).…”

“…In these studies, the basophils were also then sensitized with a gp120 peptide-specific monoclonal human IgE (hIgE) that in some experiments was also conjugated with biotin for detection by flow cytometry (calibrated to absolute IgE per basophil). In previous studies, none of the Abs used for sensitization (DNP-, gp120-or penicillin(BPO)-specific IgEs) have been observed to induce signaling without Ag present (2). However, to minimize concerns that some low level, but undetectable, signaling might occur during the sensitization, sensitization was performed at 4°C for 1 h, the cells washed once with ice-cold PAG and resuspended in cold PAGCM before rapidly warming for stimulation.…”